Labeling reagents, methods for the synthesis of such reagents and methods for the detection of biological molecules

Abstract

The present invention relates to a temperature-stable labeling reagent of formula (0): in which: R 1 represents H or an alkyl, aryl or substituted aryl group, R 2 represents a detectable marker or at least two detectable markers interlinked by at least one multimeric structure, L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, R 3 and R 4 represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2 with R=alkyl or aryl, A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, preferably 0 or 1, —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, —CH 2 S—, —Z— represents —NH—, —NHCO—, —CONH— or —O—, m is an integer between 1 and 10, preferably between 1 and 3, and p is an integer between 1 and 10, preferably between 1 and 3. The present invention also describes a method for the synthesis of said labels and also applications for the labeling of biological molecules, in particular of nucleic acids, with a labeling reagent bearing the diazomethyl function. The invention is particularly suitable for use in the field of diagnostics.

1. A temperature-stable labeling reagent of formula (0): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, the detectable label being at least one label capable of directly or indirectly generating a detectable signal, and the detectable label being selected from the group consisting of an enzyme, a chromophore, a group with an electron density detectable by electron microscopy, a group with an electron density detectable by its electrical property, a radioactive molecule, and indirect systems, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, 
 A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 —Z— represents —NH—, —NHCO—, or —CONH—, 
 m is an integer between 2 and 10, and 
 p is an integer between 1 and 10. 
 
     
     
       2. The labeling reagent according to  claim 1 , of formula (1): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 1  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, and 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 m is an integer between 2 and 10, and 
 p is an integer between 1 and 10. 
 
     
     
       3. The reagent according to  claim 2 , wherein p is less than or equal to m. 
     
     
       4. The reagent according to  claim 2 , of formula (2): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by means of at least one multimeric structure, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, and 
 q is an integer between 2 and 10. 
 
     
     
       5. The reagent, according to  claim 4 , of formula (3): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by means of at least one multimeric structure, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl. 
 
     
     
       6. The reagent according to  claim 5 , wherein R 2  consists of a D-biotin residue of formula (4): 
       
         
           
           
               
               
           
         
       
     
     
       7. The reagent according to  claim 6 , wherein R 1  is CH 3 , and R 3  and R 4  each represent H. 
     
     
       8. The reagent according to  claim 4 , in which the structure -(L) n - consists of:
 spermine or N,N′-bis(3-aminopropyl)-1,4-diaminobutane: NH 2 —(CH 2 ) 3 —NH—(CH 2 ) 4 —NH—(CH 2 ) 3 —NH 2 , or 
 spermidine or N-(3-aminopropyl)-1,4-butanediamine: H 2 N—(CH 2 ) 4 —NH—(CH 2 ) 3 —NH 2 , or 
 a derivative containing an alanine motif: NH 2 —CH 2 —CH 2 —COOH. 
 
     
     
       9. A temperature-stable labeling reagent of formula (6): 
       
         
           
           
               
               
           
         
       
       in which:
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, the detectable label being at least one label capable of directly or indirectly generating a detectable signal, and the detectable label being selected from the group consisting of an enzyme, a chromophore, a group with an electron density detectable by electron microscopy, a group with an electron density detectable by its electrical property, a radioactive molecule, and indirect systems, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, 
 A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazo function with the aromatic ring and u is an integer between 0 and 2, 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 —Z— represents —NH—, —NHCO—, or —CONH—, 
 m is an integer between 2 and 10, and 
 p is an integer between 1 and 10. 
 
     
     
       10. The labeling reagent, according to  claim 9 , of formula (7): 
       
         
           
           
               
               
           
         
       
       in which:
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 —Z— represents —NH—, —NHCO—, or —CONH—, 
 m is an integer between 2 and 10, and 
 p is an integer between 1 and 10. 
 
     
     
       11. The reagent according to  claim 1 , wherein:
 L comprises a motif —(O—CH 2 —CH 2 )—, repeated from 1 to 20 times, and 
 —Z— is —NH—, —NHCO— or —CONH—. 
 
     
     
       12. The reagent according to  claim 9 , wherein:
 L comprises a motif —(O—CH 2 —CH 2 )—, repeated from 1 to 20 times, and 
 —Z— is —NH—, —NHCO— or —CONH—. 
 
     
     
       13. A method for the synthesis of a labeling reagent according to  claim 1 , comprising the following steps:
 a) providing a label or a label precursor having a reactive function R 6 , 
 b) providing a linker arm of formula (8):
   R 7 —(Z—(CH 2 ) p ) m —R 8    
 
 
       in which:
 —Z— represents —NH—, —NHCO—, or —CONH—, 
 m is an integer between 2 and 10, 
 p is an integer between 1 and 10, 
 R 7  and R 8  represent two reactive functions which may be identical or 
 different, 
 c) reacting together the reactive function R 6  of said label or label precursor and the function R 7  the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, R 6  and R 7  being complementary, 
 d) providing a derivative of formula (9): 
 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazomethyl function with the aromatic ring and u is an integer equal to 0 or 1, and 
 R 9  represents a reactive function complementary to R 8 , 
 e) reacting together the reactive function R 9  of the derivative of formula (9) and the function R 8  of the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, 
 f) reacting the hydrazine or one of its derivatives with the ketone or aldehyde function to form a hydrazone, and 
 g) converting the hydrazone to a diazomethyl function by means of an appropriate treatment. 
 
     
     
       14. A method for the synthesis of a labeling reagent according to  claim 9 , comprising the following steps:
 a) providing a label or a label precursor having a reactive function R 6 , 
 b) providing a linker arm of formula (8):
   R 7 —(Z—(CH 2 ) p ) m —R 8    
 
 
       in which:
 —Z— represents —NH—, —NHCO—, or —CONH—, 
 m is an integer between 2 and 10, 
 p is an integer between 1 and 10, 
 R 7  and R 8  represent two reactive functions which may be identical or different, 
 c) reacting together the reactive function R 6  of said label or label precursor and the function R 7  the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, R 6  and R 7  being complementary, 
 d) providing a derivative of formula (9): 
 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl, 
 —Y—X— represents —CONH—, —NHCO—, —CH 2 O—, or —CH 2 S—, 
 A is a linker arm comprising at least one covalent double bond enabling the conjugation of the diazomethyl function with the aromatic ring and u is an integer equal to 0 or 1, and 
 R 9  represents a reactive function complementary to R 8 , 
 e) reacting together the reactive function R 9  of the derivative of formula (9) and the function R 8  of the linker arm of formula (8) in the presence of at least one coupling agent to form a covalent bond, 
 f) reacting the hydrazine or one of its derivatives with the ketone or aldehyde function to form a hydrazone, and 
 g) converting the hydrazone to a diazomethyl function by means of an appropriate treatment. 
 
     
     
       15. The method of synthesis according to  claim 13 , further comprising:
 an additional step consisting of protection of the ketone or aldehyde function of compound (9), and 
 a subsequent additional step consisting of deprotection of said ketone or aldehyde function. 
 
     
     
       16. The method of synthesis according to  claim 14 , further comprising:
 an additional step consisting of protection of the ketone or aldehyde function of compound (9), and 
 a subsequent additional step consisting of deprotection of said ketone or aldehyde function. 
 
     
     
       17. A method for the labeling of a biological molecule, comprising bringing into contact, in a homogeneous solution in a substantially aqueous buffer, the biological molecule and a reagent according to  claim 1 . 
     
     
       18. A method for the labeling of a biological molecule, comprising bringing into contact, in homogeneous solution in a substantially aqueous buffer, a biological molecule and a reagent according to  claim 9 . 
     
     
       19. A labeled biological molecule which can be obtained by the method according to  claim 17 . 
     
     
       20. A labeled biological molecule which can be obtained by the method according to  claim 18 . 
     
     
       21. A method for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid, the method comprising:
 fragmenting the nucleic acid, 
 attaching a label to at least one of the fragments by means of a labeling reagent chosen from the reagents according to  claim 1 , 
 said reagent coupling covalently and predominantly on at least one phosphate of said fragment. 
 
     
     
       22. A method for the labeling and fragmentation of a single-stranded or double-stranded nucleic acid, the method comprising:
 fragmenting the nucleic acid, 
 attaching a label to at least one of the fragments by means of a labeling reagent chosen from the reagents according to  claim 9 , 
 said reagent coupling covalently and predominantly on at least one phosphate of said fragment. 
 
     
     
       23. The method according to  claim 21 , wherein the labeling reagent is chosen from the compounds of formula (3): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, and 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl. 
 
     
     
       24. The method according to  claim 22 , wherein the labeling reagent is chosen from the compounds of formula (3): 
       
         
           
           
               
               
           
         
       
       in which:
 R 1  represents H or an alkyl, aryl or substituted aryl group, 
 R 2  represents a detectable label or at least two detectable labels interlinked by at least one multimeric structure, 
 L is a linker arm comprising a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1, and 
 R 3  and R 4  represent, independently of one another: H, NO 2 , Cl, Br, F, I, R 2 -(L) n -Y—X—, OR, SR, NR 2 , R, NHCOR, CONHR, COOR, —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 3 —CH 2 —NH—R 2 , or —CO—NH—(CH 2 ) 3 —(O—CH 2 —CH 2 ) 4 —CH 2 —NH—R 2  with R=alkyl or aryl. 
 
     
     
       25. The method according to  claim 23 , wherein the fragmentation and the labeling are carried out in two steps. 
     
     
       26. The method according to  claim 24 , wherein the fragmentation and the labeling are carried out in two steps. 
     
     
       27. The method according to  claim 23 , wherein the fragmentation and the labeling are carried out in one step. 
     
     
       28. The method according to  claim 24 , wherein the fragmentation and the labeling are carried out in one step. 
     
     
       29. The method according to  claim 25 , wherein the labeling is carried out in a substantially aqueous homogeneous solution. 
     
     
       30. The method according to  claim 27 , wherein the labeling is carried out in a substantially aqueous homogeneous solution. 
     
     
       31. The method according to  claim 26 , wherein the labeling is carried out in a substantially aqueous homogeneous solution. 
     
     
       32. The method according to  claim 25 , wherein the fragmentation is carried out by an enzymatic, physical, or chemical process. 
     
     
       33. The method according to  claim 26 , wherein the fragmentation is carried out by an enzymatic, physical, or chemical process. 
     
     
       34. A labeled nucleic acid obtained by the method according to  claim 21 . 
     
     
       35. A labeled nucleic acid obtained by the method according to  claim 22 . 
     
     
       36. A kit for the detection of a target nucleic acid, comprising a labeled nucleic acid according to  claim 34 . 
     
     
       37. A kit for the detection of a target nucleic acid, comprising a labeled nucleic acid according to  claim 35 . 
     
     
       38. A solid support to which is attached a reagent according to  claim 1 . 
     
     
       39. A solid support to which is attached a reagent according to  claim 9 . 
     
     
       40. A method for the capture of nucleic acids, comprising:
 providing a solid support to which is directly or indirectly attached at least one biological molecule according to  claim 19 , the biological molecule or the nucleic acid comprising a diazomethyl function, 
 bringing into contact a biological sample which may contain free nucleic acids, and 
 washing the solid support where the molecule(s) is (are) covalently attached at least to a nucleic acid. 
 
     
     
       41. A method for the capture of nucleic acids, comprising the following steps:
 providing a solid support to which is directly or indirectly attached at least one biological molecule according to  claim 20 , the biological molecule or the nucleic acid comprising a diazomethyl function, 
 bringing into contact a biological sample which may contain free nucleic acids, and 
 washing the solid support where the molecule(s) is (are) covalently attached at least to a nucleic acid.